RESEARCH ARTICLE Open Access

Observation of risk factors, clinical manifestationsand genetic characterization of recent NewcastleDisease Virus outbreak in West MalaysiaSeetha Jaganathan1,3,4, Peck Toung Ooi1*, Lai Yee Phang2, Zeenathul Nazariah Binti Allaudin1, Lai Siong Yip5,Pow Yoon Choo5, Ban Keong Lim5, Stephane Lemiere6 and Jean-Christophe Audonnet6

Abstract

Background: Newcastle disease virus remains a constant threat in commercial poultry farms despite intensivevaccination programs. Outbreaks attributed to ND can escalate and spread across farms and states contributingto major economic loss in poultry farms.

Results: Phylogenetic analysis in our study showed that eleven of the samples belonged to genotype VIId. Allfarms were concurrently positive with two immunosuppressive viruses; Infectious Bursal Disease Virus (IBDV) andMarek’s Disease Virus (MDV). Amino acid sequence analysis confirmed that eleven of the samples had sequencemotifs for velogenic/mesogenic strains; three were lentogenic.

Conclusion: In conclusion, no new NDV genotype was isolated from the 2011 NDV outbreak. This study suggests thatthe presence of other immunosuppressive agents such as IBD and MDV could have contributed to the dysfunction ofthe immune system of the chickens, causing severe NDV outbreaks in 2011. Risk factors related to biosecurity and farmpractices appear to have a significant role in the severity of the disease observed in affected farms.

Keywords: Newcastle disease virus, Infectious Bursal Disease, Marek’s Disease, Immunosuppressive agents,Recent outbreak, Risk factors, Phylogenetic study, Genetic characterization

BackgroundNewcastle disease (ND) is a highly contagious viral diseasein domestic poultry, aviary and wild birds. Despite intensivevaccination programs, the virus remains a constant threatto the commercial poultry farms in Malaysia [1]. Thedisease is classified in the World Organization for AnimalHealth (OIE) as a notifiable disease (formerly list A) [2, 3].It is a member of the order Mononegavirales, familyParamyxoviridae and genus Avulavirus with an envelopedvirus which has a negative-sense, non-segmented single-stranded RNA genome consisting of 15,586 nucleotides[4]. Its genome comprises six genes: nucleoprotein(NP), phosphoprotein (P), matrix protein (M), fusionglycoprotein (F), hemagglutinin-neuraminidase (HN)glycoprotein and large polymerase protein (L). Of the 6

genes found in NDV, its two membrane proteins, the Fgene and the HN gene are most important in determin-ation of its virulence. The fusion (F) protein is responsiblein mediating fusion of the viral envelope with cellularmembranes and the haemagglutinin-neuraminidase(HN) protein is involved in cell attachment and release[4–6].Newcastle disease virus strains are classified into 3

pathotypes, highly virulent (velogenic), intermediate(mesogenic) or non-virulent (lentogenic) [7]. Tradition-ally, NDV pathotypes are most commonly distinguishedby nucleotide sequencing. The consensus sequence of theF protein cleavage site of velogenic and mesogenic strainsis 112(R/K)RQ(R/K)RF117; the consensus sequence of thelentogenic F cleavage site is 112(G/E)(K/R)Q(G/E)RL117

[7, 8]. A recent finding documented that a change ofglutamine to basic residue arginine (R) at position114 of the F cleavage site reduced the viral replicationand attenuated the virus pathogenicity. The paper also

* Correspondence: ooi@upm.edu.my1Department of Clinical Studies, Faculty of Veterinary Medicine, UniversitiPutra Malaysia, 43400 UPM, Serdang, Selangor, MalaysiaFull list of author information is available at the end of the article

© 2015 Jaganathan et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Jaganathan et al. BMC Veterinary Research (2015) 11:219 DOI 10.1186/s12917-015-0537-z

reported that the pathogenicity was further reducedwhen isoleucine (I) at position 118 was substituted byvaline [9].NDV isolates have been classified into lineages or

genotypes based on the analysis of the fusion (F) gene.Aldous et al. 2003 initially used the lineage classificationsystem which grouped NDV isolates into six lineages(1–7) and 13 sub-lineages. Another lineage (lineage 7)and seven other sub-lineages were later proposed. An-other classification for NDV classifies the virus into twomajor groups called class I and class II. There are ninegenotypes (1–9) in Class I and eleven genotypes (I-XI) inClass II (2) with genotypes I, II, VI, and VII being furtherdivided into sub-genotypes 1a and 1b, II and IIa, VIathrough VIf and VIIa through VIIh [10–13]. Genotype Iconsists of the avirulent strains of NDV, while viruses ofgenotypes II, III and IV were reported to be responsiblefor the first panzootics before the 1960s [10]. GenotypeV was thought to be responsible for the second panzoo-tics in the early 1970s and genotype VII viruses causedthe third panzootics in racing pigeons during the 1980s[4, 6, 8, 14–21]. Severe outbreaks in Western Europe,South Africa and Southern Europe, Taiwan and Chinasince the early 1990s have been caused by the prevalentgenotype VII (VIIa-VIId), constituting the fourth pan-zootic of NDV [14–16, 18, 22]. Genotype VIII has beenfound to cause enzootic infections in Southern Africaand is believed to have originated from the Far East [15].Genotype IX of the ND virus has caused sporadic NDVinfections in some regions of China [2, 19], whereas Tsaiet al. first demonstrated novel genotype X viruses inTaiwan [18, 21, 23, 24].The clinical signs and pathological lesions of ND vary

with the age and species of birds, the immune status ofthe host and environmental conditions. A very highnumber of NDV cases with high mortality in broilersand lower prevalence in layers, breeders and nativebroilers were reported by field veterinarians towards theend of 2010 through 2011 in Malaysia. Newcastle diseasevirus infection was suggested as the tentative diagnosison the basis of history, clinical syndrome, gross lesions,serology as well as isolation of NDV, or the presence ofNDV by PCR and/or molecular characterization of thefusion protein gene. These cases were reported in farmswhich had various combinations of primary and boostervaccinations with lentogenic as well as mesogenic NDVvaccine, or inactivated NDV vaccines. Though vacci-nated with NDV vaccines, some of the chicken farmswere totally wiped out. Various hypotheses were raisedto explain the occurrences of the disease which included1) a change in pathogenicity of ND virus, 2) vaccineand vaccination practices, and 3) concurrent infectionwith other immunosuppression organisms or presenceof other non-infectious agents. Previous studies have

demonstrated the presence of genotype VII and geno-type II in Malaysia from 2004 until 2010 [1, 20, 25].Here we will describe the genetic characterization ofthe recent isolates from the 2011 NDV outbreaks inWest Malaysia with the objective of assessing the riskfactors associated with the disease in affected farms.This will investigate the presence of IBDV and MDVas possible immunosuppressive agent, and to deter-mine if there are any new NDV genotype in the 2011outbreak cases.

MethodsAnimals ethicsAll samples were collected under the supervision ofinstitution veterinarians. The study was conducted fol-lowing the guidelines as stated in the Code of Practicefor Care and use of Animals for Scientific Purposes asstipulated by Universiti Putra Malaysia and compliedwith the current guidelines for the care and use of ani-mals and was approved by the Institutional Animal Careand Use Committee (IACUC), Faculty of VeterinaryMedicine, Universiti Putra Malaysia. There was no ex-perimental research done on the animals. No animalswere deliberately sacrificed or injured during the sam-pling procedure. Every effort was made to minimize anydistress or unnecessary culling.

SamplingOrgan samples were collected from twelve broiler farmsdisplaying typical clinical signs for ND (Fig. 1) in variousstates in West Malaysia. Five sick birds per farm weresacrificed for the collection of specimens (trachea, lung,spleen, caecal tonsils, proventriculus, intestine, brain,liver, kidney, lymph nodes, bone marrow and bursal ofFabricius). Two farms from East Malaysia with no clin-ical ND infection were included into the study as nega-tive control.Consent was obtained from all farm owners for the

harvesting of chicken organ samples from their farms.

Observation of risk factors and clinical manifestationA standardized survey was used to assess risk factorsassociated with the high mortality observed. The surveycovered the following parameters 1) flock characteris-tics, 2) vaccination programs, 3) mortality and morbid-ity rates, 4) age of occurrence, clinical and necropsylesions and 5) farm management factors such as single/multi age farming practices, source of day old chick(DOC), breed of broilers, number of houses affected,stocking density, disease in neighboring farm, previoushistory of poor performance/mortality, measures takenin outbreak, measures taken after outbreak for nextcycle and performance of the next 2 grow-outs after

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first incidence. A scoring system (1–5) with 1 (poor) to5 (very good) was used to evaluate the biosecurity anddisinfection status of farms. The data was compiled andanalyzed using t-test, chi (χ)2 and appropriate statisticaltests to compare with the negative controls.

SerologySerum samples were collected from only four farmsfrom vaccinated flocks (25–40 days), as the study wasconducted from commercial production farms withcurrent ND outbreaks. It was not possible to obtainpaired serum samples for comparison from every farmas the severity of the outbreak caused very high mortal-ity. The ND-HI titer analysis was conducted by Vet FoodAgro Diagnostics (M) Sdn. Bhd.

Screening of NDV, IBD, MDVOrgan samples were pooled for screening by real-timePCR for NDV and for other immunosuppressive agentsi.e., MDV and IBDV. The organ samples were subjectedto nucleic acid extraction by using the Trizol LS reagent(Invitrogen, USA) following the standard manufacturer’sprotocol. The real time PCR was established frommethods previously described [1, 26, 27]. The realtime PCR mixtures consisted of 10 μl of SYBR greenmaster mix, the respective primer sets and PCR gradewater to make up the final volume of 20 μl per reac-tion. The PCR mixtures were subjected to real timePCR amplification in a 384-well microplate in theLC480 Real Time PCR instrument (LC 480, Roche).The melting peaks and melting curves were observed

Fig. 1 Clinical signs observed from the outbreak. a A typical torticollis is shown. These symptoms normally occur 7 to 10 days after a complaint of highmortality is reported. b In severely affected birds, mild swollen head and dyspnea with profuse secretions in the trachea were found. c & d Hemorrhagic &necrosis of intestines especially the caecal tonsils & peyer’s patches were found. e Upon PM, the trachea was severely congested and late in the diseasestages pericarditis, perihepatitis and caseous air sacculitis were observed. f Proventicular hemorrhages were consistent. g, h & i Bursa atrophy was alsocommonly found in the outbreak. The cut surfaces of the bursa were hemorrhagic – quite atypical from ND infection which prompted us to look forother infectious agents. Not shown above was atrophic thymus

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by using the Absolute Quant Software provided withthe instrument. The respective primer sets are as follow,NDV primers specific for the fusion protein gene, 5′-ATGGGC(C/T) CCA GA(C/T) CTT CTA C-3′ (forward)and 5′-CTG CCA CTG CTA GTT GTG ATA ATC C-3′(reverse) (Amplicon size: 545 bp); IBD primers, 5′-GTRAC RAT CAC ACT GTT CTC AGC-3′ (Y=C/T);(R=A/G) (forward) and 5′-GAT GTR AYT GGC TGGGTT ATC TC-3′ (reverse) (Amplicon size: 248 bp);MDV-serotype 1 primers, 5′-GAC TCG CTC GCA CATC-3′ (forward) and 3′-CGA CAC TCC GCA GTT-5′ (re-verse) (Amplicon size: 102 bp); MDV-serotype 2 primers;5′-GTT TCG TCT ACC ACC CG-3′ (forward) and 5′-ATG CCA CTG TAT TTG ATC TCC-3′ (reverse)(Amplicon size: 139 bp); MDV-serotype 3 primers, 5′-ACC GCA ACT CTT CTC ACA-3′ (forward) and 3′-CTC GGG CAA CCT CTA CAT-5′ (reverse) (Ampliconsize: 201 bp). Primer sets for MDV serotype 1, 2 and 3were designed by using the Primer Premier 5 softwarefrom Premier Biosoft.

Sequencing, amino acid sequence analysis, phylogeneticconstruction of NDV from the 2011 outbreakThe same primer sets for amplifying NDV as describedabove [1, 26] were used for sequencing. The PCRproducts of the expected amplicon sizes were purifiedby using the PCR clean-up gel extraction kit accordingto the manufacturer’s protocol with slight modifications(Analytik Jena, Germany). Sequencing of the fusionprotein gene of NDV from the 14 farms was done in acommercial sequencing facility using the BigDye®Terminator v3.1 cycle sequencing kit. In order to con-firm that all positive cases were true Newcastle diseasevirus, a Basic Local Alignment Search Tool (BLAST)search of the sequence was done in the Genbank®database (Data not shown). The sequence editing andassembly were done by using BioEdit® SequenceAlignment Editor version 7.0.5.2 (Tom Hall, US). Se-quences were aligned by using ClustalX™. The phylo-genetic tree was constructed by using the distance-based neighbor joining method by using Mega™ 5software (Biodesign Institute, Tempe, Arizona) andevaluated using the bootstrapping method calculatedon 1000 repeats of the alignment. The sequenceidentity matrix was generated with BioEdit® SequenceAlignment Editor version 7.0.5.2 (Tom Hall, US). Allsequences used for constructing the phylogenetic treeare listed in Table 1.

Results & discussionObservation of risk factors and clinical manifestationsThe results of the survey are shown in Tables 2, 3, 4 and5. Table 2 describes farm characteristics and type ofbreeds used. All farms were multi-age broiler farms

stocked with various commercial breeds namely Cobb,Ross or commercial native birds. The mean flock popu-lation was about 90,000 broilers (83.3 % of houses wereopen sided houses). On average, 7.2 houses were af-fected. Over 90 % of the farms surveyed were locatedwithin 1 km radius to other broiler and reported highmortality in neighboring farms. More than 50 % of thefarms had poor performance in its two previousgrow-outs (P < 0.05), reported that the day-old-chickquality were poor in the affected flock (P < 0.01) and hadunsubstantiated evidence that the chicks were sourcedfrom breeder flocks which had been reported withhigh mortalities resembling ND (P < 0.05). The averageage of onset of disease was 15.9 (P < 0.05) days oldwhich was reported to be at the time or after IBD vac-cinations were administered and 28 days in the nega-tive control farms.The frequency of vaccination in the 12 study farms

for Newcastle disease, Infectious Bronchitis, InfectiousBursal disease, Marek’s disease and Swollen HeadSyndrome (SHS); a disease caused by Avian metap-neumovirus; were 100 %, 100 %, 91.7 %, 8.3 % and8.3 % respectively (Table 4). The frequency of NDvaccination for lentogenic, mesogenic and inactivatedvaccines were 100 %, 33.3 % and 50.0 % with allfarms practicing multiple ND vaccinations in the life-cycle of the broilers. Biosecurity and farm sanitationscores were 1.7 and 2.6 respectively.The onset of clinical ‘ND’ disease was reported at

15.9 days old (P < 0.05) with a final grow out mortality ofaffected flocks at 32.3 % (P < 0.05) (Table 5). All the farmsinitiated treatment with antimicrobials and supportivetreatments (vitamins and electrolyte) and upgradedsanitation and disinfection practices when the disease wasobserved or when advised by field veterinarians. Theprimary clinical signs were inappetance, depression, ruf-fled feathers, whitish to greenish and watery diarrheoa,head swelling and dyspnoea. Torticollis was seen at about7–10 days after the onset of clinical signs. Gross pathologylesions included profuse fluids in the trachea and bron-chus, congested trachea, gizzard and proventricular ero-sions and hemorrhages, hemorrhages on Peyer’s patchesand caecal tonsils and in other visceral organs. Bursa (P <0.05) and thymus atrophy were present in the majority ofcases. The disease process appeared to be of acute onsetwith significantly reduced observations of air sacculitis,perihepatitis and peritonitis (P < 0.05). NDV, IBDV andMDV were detected in organs and tissues at 100 %, 83 %and 83 % respectively by PCR, with MD Serotypes 2, 1and 3 in descending order of detection. NDV, IBDV andMDV were detected concurrently in 75 % of farms(Table 6). The concurrent presence of IBDV and MDVwith NDV were significant at P < 0.05 and with the pres-ence of MDV Serotype 1 (P < 0.01).

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SerologySerology results from one farm (Farm 6) showed thatthe antibody titer was low and not as expected with amean titer of 1.8. Antibody titer from three other farms(Farm 4, 7 and 8) was very high for a broiler which indi-cates that there’s an infection.

Screening for NDV, IBD and MDVSamples from all farms were found to be positive for NDV,IBD and MDV, and clearly evident that immunosupressioncontinues to be a major problem for the poultry industry.IBDV is one of the most common immunosuppressiveagents in poultry. It primarily targets the bursa of Fab-ricious which is committed to the differentiation and pro-liferation of B-lymphocytes into antibody-producingplasma cells. In the bursa, IBDV produces severe de-struction of B-lymphocytes by either necrosis or apop-tosis, and consequently the antibody-mediated response(humoral) is affected [27, 28]. Recent studies have alsodemonstrated that this virus can also hinder some of themechanisms of cellular immunity making chickens moresusceptible to viral respiratory infections and elevatingmortality. Thus, the immune response to vaccines isimpaired and the overall productive performance may besignificantly decreased in all types of chickens. Similarly,Marek’s Disease, which is an ubiquitous, complex, lym-phoproliferative disease of chickens caused by a strongcell-associated alpha herpesvirus, MD virus (MDV) is

Table 2 Comparison of serology titer from the surveyed NDoutbreak vs. serology titer from non-survey farms ND outbreak

GMT Mean

Farm 6 (survey) ND outbreak with IBD andMD detected

1.5 1.8

Farm 4, 7, 8 (survey) ND outbreak with IBD andMD detected

212.8 390.4

Table 1 NDV isolates derived from this study and other isolatesreported previously

No Isolate name Genbank®accessionnumber

Genotype Country Reference

1 V4Queensland

M24693 I Australia Genbank®

2 Ulster/67 M24694 I N. Ireland Genbank®

3 F7 JN613118 I Malaysia This study

4 Lasota M24696 II USA Genbank®

5 MB061/07 GQ901891 II Malaysia Genbank®

6 F5 JN613116 II Malaysia Genbank®

7 F6 JN613117 II Malaysia Genbank®

8 Miyadera M24701 III Japan Genbank®

9 Italien EU293914 IV Italy Genbank®

10 Herts/33 AY741404 IV UK Genbank®

11 CA1085/71 AF001106 V USA Genbank®

12 H-10/72 AF001107 V Hungary Genbank®

13 TX3503/04 EU477190 VI USA Genbank®

14 NDV05-027 DQ439885 VI China Genbank®

15 Q-GB 506/97 AF109887 VI UK Genbank®

16 DK-1/95 AF001129 VI Denmark Genbank®

17 Iraq AG68 AF001108 VI Iraq Genbank®

18 Lebanon-70 AF001110 VI Lebanon Genbank®

19 MB047/05 GQ901895 VIIa Malaysia Genbank®

20 Cockatoo/14698/90

AY288998 VIIa Indonesia Genbank®

21 ZA360/95 AF109876 VIIb S. Africa Genbank®

22 ZW3422/95 AF109877 VIIb Zimbabwe Genbank®

23 NDV05-055 DQ439910 VIIc China Genbank®

24 TW/2000 AF358786 VIIc/d Taiwan Genbank®

25 F8 JN613119 VIId Malaysia Genbank®

26 Ch/2000 AF358788 VIId China Genbank®

27 MB064/05 GQ901893 VIId Malaysia Genbank®

28 MB016/07 GQ901894 VIId Malaysia Genbank®

29 F1 JN613112 VIId Malaysia This study

30 F2 JN613113 VIId Malaysia This study

31 F3 JN613114 VIId Malaysia This study

32 F4 JN613115 VIId Malaysia This study

33 F9 JN613120 VIId Malaysia This study

34 F10 JN613121 VIId Malaysia This study

35 F11 JN613122 VIId Malaysia This study

36 F12 JN613123 VIId Malaysia This study

37 F13 JN613124 VIId Malaysia This study

38 F14 JN613125 VIId Malaysia This study

39 MB043/06 GQ901896 VIId Malaysia Genbank®

40 MB091/05 FJ008916 VIId Malaysia Genbank®

41 MB093/05 FJ008917 VIId Malaysia Genbank®

Table 1 NDV isolates derived from this study and other isolatesreported previously (Continued)

42 MB095/05 FJ008918 VIId Malaysia Genbank®

43 MB128/04 FJ008923 VIId Malaysia Genbank®

44 DE143/95 AF109881 VIId UK Genbank®

45 TW/95-1 AF083960 VIIe Taiwan Genbank®

46 MB076/05 GQ901892 VIIe Malaysia Genbank®

47 AF2240 AF048763 VIII Malaysia Genbank®

48 MB085/05 GQ901901 VIII Malaysia Genbank®

49 QH-1/79 AF378250 VIII China Genbank®

50 QH-4/85 AF378252 VIII Malaysia Genbank®

51 ZhJ-1/85 AF458023 IX Malaysia Genbank®

52 FJ-1/85 AF458009 IX Malaysia Genbank®

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progressive in nature with a relatively long incubationperiod and the virulent virus can remain in the host with-out producing any clinical syndromes [23, 24, 29, 30].The primary cell targets of MDV infection are lympho-cytes, and as a result, early effects are mainly seen inlymphoid organs such as the bursa of Fabricius (source ofB lymphocytes), the thymus (primary source of T lym-phocytes) and the spleen. Consequences of these MDV-induced immunosuppresion have also shown to cause re-duced resistance to other concurrent infections [29–31].Therefore, based on the findings, the presence of IBDand MDV suggest a significant negative impact on theimmune system and growth of these broiler chickens.NDV itself was not immunosuppressive, and vaccinationwith killed-NDV vaccines failed to reduce incidences ofNDV outbreaks, because the core underlying factors suchas IBD and MDV that were causing sub-clinical infectionswere not addressed, thus reducing the protective effect ofthe NDV vaccination programs.

Sequencing, amino acid sequence analysis, phylogeneticconstructionBLAST® analysis showed that all samples were true NDVcases when compared with other sequences on Genbank®.Amino acid sequence analysis of the 535 bp fragment ofthe fusion protein (F) gene of the fourteen MalaysianNDV isolates showed that eleven of the isolates werecategorized as velogenic virus and three were lentogenic.The eleven velogenic strain had the F cleavage site motif112R-R-R-K-R-F117 while two of the lentogenic strainhad the F cleavage site motif 112G-R-Q-G-R-L117, whilstone sequence had the F cleavage site motif 112G-K-Q-G-R-L117 at the C-terminus of the F2 protein and phenyl-alanine (F) residue at amino acid position 117 of the N-terminus of the F1 protein (Table 7). Phylogenetic analysisrevealed that 11 of the Malaysian isolates clustered closelywith the genotype VIId strains, one of the Malaysianisolate grouped together with genotype I and two of theisolates grouped with genotype II (Fig. 2). Of the elevenMalaysian isolates that grouped to form genotype VIId,ten (F1, F2, F3, F4, F9, F10, F11, F12, F13, F14) had

Table 5 Zootechnical results, clinical and necropsy findings

Risk factor Affectedfarms

Negativecontrols

Age of first occurrence of disease 15.9a 28.0a

Age of clinical and necropsy examination 24.8a 32.5a

Mortality at grow-out, % 32.3 %a 4.5 %b

Presence of respiratory disease 100 %a 100 %a

Presence of enteric disease 100 %a 0 %b

Torticolis and neurological signs 91.7 %a 0 %b

Haemorrhages in more than 1 visceral organ 83.3 %a 0 %b

Thymus atrophy 100 %a 0 %b

Bursal atrophy 75 %a 50 %b

Ascites 8.3 %a 50 %a

Air-sacculitis, perihepatitis and peritonitis 16.7 %a 50 %b

Clouded air sacs 50 %c 100 %d

Note: a,bValues in different columns bearing different superscripts aresignificantly different (P < 0.05), c,dValues in different columns bearing differentsuperscripts are significantly different (P < 0.01)

Table 6 Detection of infectious agents concurrently with NDVpositive samples

Presence of concurrent infectious agent indisease farms

Frequency of detection

IBD 83.0 %

MD 83.0 %

IBD+MD 75.0 %

MD Serotype 1 58.0 %

MD Serotype 2 75.0 %

MD Serotype 3 67.0 %

Table 4 Vaccination and assessment of biosecurity andsanitation status

Risk factor Affected farms Negative controls

Biosecurity status 1.7a 2.5a

Sanitation and disinfection status 2.6a 2.5a

Vaccination with NDv 100 %a 100 %a

Vaccination with IBv 100 %a 100 %a

Vaccination with IBDv 91.7 %a 91.7 %a

Vaccination with MDv 8.3 %a 0 %b

Vaccination with SHSv 8.3 %a 0 %b

Note: a,bValues in different columns bearing different superscripts aresignificantly different (P < 0.05), c,dValues in different columns bearing differentsuperscripts are significantly different (P < 0.01)

Table 3 Farm characteristics and history

Risk factor Affectedfarms

Negativecontrols

No of farms 12 2

Number of broilers per farm 89,950a 87,500a

Number of houses per farm 11.1a 6.5a

Number of houses affected per farm 7.2a 1.5a

Type of management – multi-age Multi-age Multi-age

Type of housing – open-sided housing Open Open

Farms within 1 km of affected farm 92 %a 50 %a

Poor performance in the last 2 grow outs 50 %c 0 %d

Neighboring farms with history of disease 91.7 %a 0 %b

Complaints of poor day-old-chick quality 50 %c 50 %d

Disease after IBD vaccination or about Week 2-3 83.3 %a 50 %b

Suspected source of chicks from breeder farmswith disease

66.7 %a 0 %b

Note: a,bValues in different columns bearing different superscripts aresignificantly different (P < 0.05), c,dValues in different columns bearing differentsuperscripts are significantly different (P < 0.01)

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between 97.9 to 98.7 % sequence identity similarities withother Malaysian isolates responsible for the Newcastledisease outbreaks in 2004–2005 and 2007 which werepreviously reported by researchers from Universiti PutraMalaysia [20, 28, 31]. All these isolates have between 91and 92 % similarities with the Indonesian strain (cocka-too/14698/90). One (F8) of the Malaysian isolate whichgrouped with genotype VIId had 96.1 % similarities withthe China strain (Ch/2000). Of the three lentogenic strainsisolated from this study, two (F5, F6) had between 97.4and 97.5 % nucleotide sequence similarities with genotypeII while one isolate (F7) had around 88.8 % nucleotidesequence similarities with genotype I.Overall, based on the amino acid sequence analysis,

eleven of the farms displayed the typical sequencemotifs for velogenic/mesogenic, while three had thesequence motif for lentogenic strains. This correlateswith the fact that two of the samples (F6 & F7) werecollected as negative controls from farms with nomortality. Based on phylogenetic investigations, eleven(F1, F2, F3, F4, F8, F9, F10, F11, F12, F13, F14) ofthe samples clustered with the genotype VIId and hada very close relationship with the previously isolatedMalaysian isolates (MB043/06, MB091/06, MB093/05,MB095/05, MB128/04) which suggest that similarNDV strains have been circulating in this region forseveral years now [1, 20, 25].

Nucleotide sequence accession numbersThe complete genomic sequences of the 14 NDV iso-lates reported in this paper were deposited with theGenBank® database under accession numbers JN613112,

JN613113, JN613114, JN613115, JN613116, JN613117,JN613118, JN613119, JN613120, JN613121, JN613122,JN613123, JN613124 and JN613125.These sequences are downloadable from Entrez™ Pop

Set data as a group of sequences.

ConclusionThe present study confirms that similar velogenic NDVgenotype VIId, as reported previously, were detected inthe study farms in spite of vaccination. In addition, nonew genotype of ND virus was found based on the gen-etic characterization. IBDV and MDV were also concur-rently detected by PCR. Immunosuppressive agents playa significant role in vaccination failures. However, therole, interactions and effect of these immunosuppressiveagents as well as other concurrent infectious and noninfectious agents not studied in the disease processcould not be fully ascertained. Risk factors such asmulti-age production practices, close proximity of farms,biosecurity and sanitation practices appear to have a rolein the outcome of the disease, in terms of severity,mortality, clinical and pathological findings. Preventivemeasures taken post outbreak such as improved bio-security and sanitation appear to have mediated im-proved performance in subsequent grow-outs. Thefindings from this study suggest that there may notbe a need for a new vaccine as the same genotypethat has been present for a long time is still respon-sible for the current ND outbreaks. Further to that,the findings also suggest that risk factors related tobiosecurity and farm practices appear to have a sig-nificant role in the severity of the disease observed

Table 7 The F cleavage site and it’s pathotypes from the Malaysian isolates

Isolate Genbank® accession no F cleavage site Genotype Pathotype Source

F1 JN613112 RRRKRF VIId Velogenic/Mesogenic This study

F2 JN613113 RRRKRF VIId Velogenic/Mesogenic This study

F3 JN613114 RRRKRF VIId Velogenic/Mesogenic This study

F4 JN613115 RRRKRF VIId Velogenic/Mesogenic This study

F5 JN613116 GRQGRL II Lentogenic This study

F6 JN613117 GRQGRL II Lentogenic This study

F7 JN613118 GKQGRL I Lentogenic This study

F8 JN613119 RRRKRF VIId Velogenic/Mesogenic This study

F9 JN613120 RRRKRF VIId Velogenic/Mesogenic This study

F10 JN613121 RRRKRF VIId Velogenic/Mesogenic This study

F11 JN613122 RRRKRF VIId Velogenic/Mesogenic This study

F12 JN613123 RRRKRF VIId Velogenic/Mesogenic This study

F13 JN613124 RRRKRF VIId Velogenic/Mesogenic This study

F14 JN613125 RRRKRF VIId Velogenic/Mesogenic This study

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in affected farms. If those factors are alleviated, theseverity of the ND problems in farms would be greatlyreduced.

AbbreviationsNDV: Newcastle disease virus; IBDV: Infectious bursal disease virus;MDV: Marek’s disease virus; PCR: polymerase chain reaction; MDV: Marek’sDisease Virus; SHS: Swollen Head Syndrome.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsSJ participated in the conceptual aspect of the work, conceived the research,performed the experiments and wrote the manuscript. OPT, PLY, ZNA, YLS,CPY, LBK, SL, JCA provided consultation and coordination. All authors readand approved the final manuscript.

AcknowledgementsThe authors would like to thank Vet Food Agro Diagnostics (M) Sdn. Bhd forproviding the samples for this study and Rhone Ma Malaysia Sdn. Bhd. andMerial for funding the research.

DisclaimerThis document is provided for scientific purposes only. Any reference to abrand or trademark herein is for informational purposes only and is notintended for a commercial purpose or to dilute the rights of the respectiveowner(s) of the brand(s) or trademark(s).

Author details1Department of Clinical Studies, Faculty of Veterinary Medicine, UniversitiPutra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia. 2Department ofBiotechnology, Faculty of Biotechnology & Molecular Science, Universiti PutraMalaysia, 43400, UPM, Serdang, Selangor, Malaysia. 3Asia-Pacific SpecialNutrients Sdn. Bhd, Lot 18B, Jalan 241, Section 51A, 46100 Petaling Jaya,Selangor, Malaysia. 4Vet Food Agro Diagnostic Sdn. Bhd., Lot 18B, Jalan 241,Section 51A, 46100 Petaling Jaya, Selangor, Malaysia. 5Rhone Ma Malaysia (M)Sdn Bhd, Lot 18B, Jalan 241, Section 51A, 46100 Petaling Jaya, Selangor,Malaysia. 6Merial S.A.S., Bio R&D, 254, Rue Marcel Merieux, 69007 Lyon,France.

Received: 6 March 2015 Accepted: 6 August 2015

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